Infection of RANKL-Primed RAW-D Macrophages with Porphyromonas gingivalis Promotes Osteoclastogenesis in a TNF-α-Independent Manner

Infection of macrophages with bacteria induces the production of pro-inflammatory cytokines including TNF-α. TNF-α directly stimulates osteoclast differentiation from bone marrow macrophages in vitro as well as indirectly via osteoblasts. Recently, it was reported that bacterial components such as LPS inhibited RANKL-induced osteoclastogenesis in early stages, but promoted osteoclast differentiation in late stages. However, the contribution to osteoclast differentiation of TNF-α produced by infected macrophages remains unclear. We show here that Porphyromonas gingivalis, one of the major pathogens in periodontitis, directly promotes osteoclastogenesis from RANKL-primed RAW-D (subclone of RAW264) mouse macrophages, and we show that TNF-α is not involved in the stimulatory effect on osteoclastogenesis. P. gingivalis infection of RANKL-primed RAW-D macrophages markedly stimulated osteoclastogenesis in a RANKL-independent manner. In the presence of the TLR4 inhibitor, polymyxin B, infection of RANKL-primed RAW-D cells with P. gingivalis also induced osteoclastogenesis, indicating that TLR4 is not involved. Infection of RAW-D cells with P. gingivalis stimulated the production of TNF-α, whereas the production of TNF-α by similarly infected RANKL-primed RAW-D cells was markedly down-regulated. In addition, infection of RANKL-primed macrophages with P. gingivalis induced osteoclastogenesis in the presence of neutralizing antibody against TNF-α. Inhibitors of NFATc1 and p38MAPK, but not of NF-κB signaling, significantly suppressed P. gingivalis-induced osteoclastogenesis from RANKL-primed macrophages. Moreover, re-treatment of RANKL-primed macrophages with RANKL stimulated osteoclastogenesis in the presence or absence of P. gingivalis infection, whereas re-treatment of RANKL-primed macrophages with TNF-α did not enhance osteoclastogenesis in the presence of live P. gingivalis. Thus, P. gingivalis infection of RANKL-primed macrophages promoted osteoclastogenesis in a TNF-α independent manner, and RANKL but not TNF-α was effective in inducing osteoclastogenesis from RANKL-primed RAW-D cells in the presence of P. gingivalis

Calcium sparks enhance the tissue fluidity within epithelial layers and promote apical extrusion of transformed cells

In vertebrates, newly emerging transformed cells are often apically extruded from epithelial layers through cell competition with surrounding normal epithelial cells. However, the underlying molecular mechanism remains elusive. Here, using phospho-SILAC screening, we show that phosphorylation of AHNAK2 is elevated in normal cells neighboring RasV12 cells soon after the induction of RasV12 expression, which is mediated by calcium-dependent protein kinase C. In addition, transient upsurges of intracellular calcium, which we call calcium sparks, frequently occur in normal cells neighboring RasV12 cells, which are mediated by mechanosensitive calcium channel TRPC1 upon membrane stretching. Calcium sparks then enhance cell movements of both normal and RasV12 cells through phosphorylation of AHNAK2 and promote apical extrusion. Moreover, comparable calcium sparks positively regulate apical extrusion of RasV12-transformed cells in zebrafish larvae as well. Hence, calcium sparks play a crucial role in the elimination of transformed cells at the early phase of cell competition

Characterization and identification of subpopulations of mononuclear preosteoclasts induced by TNF-α in combination with TGF-β in rats.

Osteoclasts are unique multinucleated cells formed by fusion of preosteoclasts derived from cells of the monocyte/macrophage lineage, which are induced by RANKL. However, characteristics and subpopulations of osteoclast precursor cells are poorly understood. We show here that a combination of TNF-α, TGF-β, and M-CSF efficiently generates mononuclear preosteoclasts but not multinucleated osteoclasts (MNCs) in rat bone marrow cultures depleted of stromal cells. Using a rat osteoclast-specific mAb, Kat1, we found that TNF-α and TGF-β specifically increased Kat1(+)c-fms(+) and Kat1(+)c-fms(-) cells but not Kat1(-)c-fms(+) cells. Kat1(-)c-fms(+) cells appeared in early stages of culture, but Kat1(+)c-fms(+) and Kat1(+)c-fms(-) cells increased later. Preosteoclasts induced by TNF-α, TGF-β, and M-CSF rapidly differentiated into osteoclasts in the presence of RANKL and hydroxyurea, an inhibitor of DNA synthesis, suggesting that preosteoclasts are terminally differentiated cells. We further analyzed the expression levels of genes encoding surface proteins in bone marrow macrophages (BMM), preosteoclasts, and MNCs. Preosteoclasts expressed itgam (CD11b) and chemokine receptors CCR1 and CCR2; however, in preosteoclasts the expression of chemokine receptors CCR1 and CCR2 was not up-regulated compared to their expression in BMM. However, addition of RANKL to preosteoclasts markedly increased the expression of CCR1. In contrast, expression of macrophage antigen emr-1 (F4/80) and chemokine receptor CCR5 was down-regulated in preosteoclasts. The combination of TNF-α, TGF-β, and M-CSF induced Kat1(+)CD11b(+) cells, but these cells were also induced by TNF-α alone. In addition, MIP-1α and MCP-1, which are ligands for CCR1 and CCR2, were chemotactic for preosteoclasts, and promoted multinucleation of preosteoclasts. Finally, we found that Kat1(+)c-fms(+) cells were present in bone tissues of rats with adjuvant arthritis. These data demonstrate that TNF-α in combination with TGF-β efficiently generates preosteoclasts in vitro. We delineated characteristics that are useful for identifying and isolating rat preosteoclasts, and found that CCR1 expression was regulated in the fusion step in osteoclastogenesis

Expression of osteoclast signaling proteins in osteoclastogenesis in RANKL-primed RAW-D cells induced by infection.

<p>RAW-D cells were stimulated with or without RANKL (50 ng/ml) or <i>P. gingivalis</i> for 22 h. RANKL-primed RAW-D cells were then retreated with or without RANKL (50 ng/ml) or <i>P. gingivalis</i> for 24 h. Total RNA was prepared, cDNA was synthesized, and real-time PCR analysis was performed using NFATc1 (A), c-fos (B), or IFNβ (C) Taqman probes. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared with unprimed control or RANKL-primed control.</p

RANKL but not TNF-α promotes osteoclastogenesis in RANKL-primed RAW-D cells in the presence of <i>P. gingivalis</i>.

<p>RAW-D cells were stimulated with RANKL (50 ng/ml) for 22 h and then re-stimulated with RANKL or TNF-α in the absence (A) or presence (B) of live <i>P. gingivalis.</i> The culture was stained for TRAP activity after 48 h of retreatment, and TRAP-positive MNCs were counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, compared to cultures without RANKL or TNF-α. (C) Expression of mRNAs for p55 and p75 TNF receptors, TLR2, and TLR4 in RAW-D cells treated with or without <i>P. gingivalis</i> for 22 h, or RANKL-primed RAW-D cells retreated with or without RANKL or <i>P. gingivalis</i> for 24 h. Total RNA was isolated, and mRNA expression was assessed by semi-quantitative RT-PCR using specific primers as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038500#s4" target="_blank">Materials and Methods</a></i>.</p

<i>P. gingivalis</i> induces osteoclastogenesis in RANKL-primed RAW-D cells in the absence of TNF-α.

<p>Analysis of TNF-α mRNA expression (A) or production of TNF-α protein (B) by <i>P. gingivalis</i> infected RANKL-primed RAW-D cells and unprimed cells. (C) Effect of neutralizing antibody against mouse TNF-α on osteoclast formation in RANKL-primed RAW-D cells induced by TNF-α or live <i>P. gingivalis</i>. RAW-D cells were primed with RANKL (50 ng/ml) for 22 h and then retreated with TNF-α or live <i>P. gingivalis</i> in the presence or absence of neutralizing antibody against mouse TNF-α or control IgG. After 24 h, RNA was extracted, and TNF-α mRNA expression was assessed by real-time PCR. After 48 h, cell supernatants were collected and analyzed for TNF-α by ELISA. After 48 h, the culture was stained for TRAP, and the number of TRAP-positive MNCs was counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared with unprimed infected RAW-D or RANKL-primed uninfected control (A), unprimed control (B), or control IgG1 (C).</p

TLR4 is not involved in osteoclastogenesis in RANKL-primed RAW-D cells induced by infection with <i>P. gingivalis</i>.

<p>Effect of <i>E.coli</i> LPS or Pam3CSK4 (A) or <i>P. gingivalis</i> LPS (B) on osteoclastogenesis in RANKL-primed RAW-D cells. (C) Effect of heat treatment of <i>P. gingivalis</i> on osteoclastogenesis in RANKL-primed RAW-D cells. (D) Effect of polymyxin B on osteoclast formation in RANKL-primed RAW-D cells induced by <i>E. coli</i> LPS, Pam3CSK4, live <i>P. gingivalis</i>, or <i>P. gingivalis</i> LPS. RAW-D cells were primed with RANKL (50 ng/ml) for 22 h and then treated with <i>E. coli</i> LPS (100 ng/ml), Pam3CSK4 (100 ng/ml), <i>P. gingivalis</i> LPS (10 µg/ml) or live <i>P. gingivalis</i> (m.o.i.  = 10) in the presence of various concentrations of polymyxin B. After 48 h, the culture was stained for TRAP, and the number of TRAP-positive MNCs was counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01 compared to untreated controls (A, B, and C) or controls without polymyxin B (D).</p

Infection of RANKL-primed RAW-D macrophages with <i>P. gingivalis</i> induces osteoclastogenesis.

<p>(A) (B) <i>P. gingivalis</i> infection of RANKL-primed RAW-D cells induces the formation of TRAP-positive MNCs. (C) <i>P. gingivalis</i> infection of RANKL-primed RAW-D cells induces mRNA expression of the osteoclast-specific gene, cathepsin K. Total RNA was isolated, and cathepsin K expression was assessed by real-time PCR. Expression levels were normalized to GAPDH. Effect of pretreatment (D), or OPG (E) on osteoclastogenesis induced by infection with <i>P. gingivalis</i>. RAW-D cells were primed with RANKL (50 ng/ml) or TNF-α (10 ng/ml) for 22 h, then infected with <i>P. gingivalis</i>, and cultured for 24–48 h. After 24 h, RNA was extracted and analyzed for gene expression. After 48 h, the culture was stained for TRAP, and TRAP-positive MNCs were counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared to uninfected control (B, C) or untreated control (D).</p

Infection of RANKL-primed BMM with <i>P. gingivalis</i> induces osteoclastogenesis in the absence of TNF-α.

<p>(A) Infection of RANKL-primed BMM with <i>P. gingivalis</i> induces osteoclastogenesis. Representative photographs are shown. (B) Effect of neutralizing antibody against mouse TNF-α on osteoclastogenesis in RANKL-primed BMM induced by TNF-α or live <i>P. gingivalis</i>. BMM were stimulated with RANKL (50 ng/ml) for 22 h and then re-stimulated by TNF-α or live <i>P. gingivalis</i> (m.o.i.  = 10) in the presence or absence of neutralizing antibody against mouse TNF-α or control IgG. At the end of culture, the culture was stained for TRAP, and TRAP-positive MNCs were counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, compared with control IgG1.</p

Possible role of TNF-α in osteoclastogenesis in the presence or absence of <i>P. gingivalis</i>.

<p>Macrophages respond to infection with <i>P. gingivalis</i> by producing TNF-α, which stimulates osteoclastogenesis in osteoclast precursor cells in the absence of <i>P. gingivalis</i> (A). However, osteoclast precursor cells primed with RANKL do not produce TNF-α and respond differentially to various stimuli. (B) Cells that are not stimulated do not differentiate into osteoclasts. (C) Cells that are continuously re-stimulated with RANKL differentiate into osteoclasts in an NFATc1- and NF-κB-dependent manner in the presence of <i>P. gingivalis</i>. (D) Cells that are infected with <i>P. gingivalis</i> differentiate into osteoclasts in an NFATc1-dependent and NF-κB-independent manner. TNF-α does not stimulate osteoclastogenesis in osteoclast precursor cells in the presence of <i>P. gingivalis</i>, whereas RANKL stimulates osteoclastogenesis in the presence or absence of <i>P. gingivalis</i>.</p